The mechanisms underlying the persistence of HCV infection are poorly understood. HCV E2 and NS5A have been suggested to inhibit cellular antiviral responses; however, data have been controversial. In collaboration with Dr. Michael Gale's group, we recently demonstrated that the NS3/4A serine protease blocks virus-induced phosphorylation and activation of interferon regulatory factor 3 (IRF-3), a key transcriptional factor in initiating cellular antiviral responses. However, the detailed consequences of this blockade, i.e. the alterations of downstream hepatocellular antiviral defensive gene expression, which may contribute to the persistence of hepatitis C, remain to be completely elucidated. In addition, the mechanisms by which NS3/4A protease blocks IRF-3 phosphorylation are yet not known. The primary goal of this proposal is to explore the answers of these issues using the recently-developed functional genomics approaches including microarrays and proteomics. Specific Aim 1, to identify and profile IRF-3 dependent and independent antiviral response genes of hepatocytes that are blocked /suppressed by NS3/4A expression or by genome-length HCV RNA replication. Cells that (1) conditionally express the NS3/4A protease, (2) contain replicating full-length HCV RNA, or (3) conditionally express a dominant negative IRF-3 mutant will be challenged with Sendai virus (SenV), and differences in SenV-activated gene expression will be identified using Affymetrix microarrays and compared with those from clonally matched HCV-negative cells. These experiments should identify antiviral genes downstream of the IRF-3 pathway and other possible pathways that are blocked/suppressed by NS3/4A protease. They will also provide information on whether other HCV proteins and IRF-3 independent pathways contribute to the persistence of HCV infection. Specific Aim 2, to explore the cellular changes at protein level by which NS3/4A protease blocks IRF-3 phosphorylation using a proteomics approach. Cells that conditionally express the NS3/4A protease will be challenged with SenV, and differences in protein expression before and after challenge, in the presence and absence of NS3/4A will be identified by two-dimensional gel eletrophoresis coupled with automated polypeptide sampling and polypeptide sequencing by mass spectrometry. This approach is likely to identify the virus-activated kinase (VAK) or other signaling components indispensable for virus-induced IRF-3 phosphorylation that are inhibited by the NS3/4A serine protease. This proposal should lead to a better understanding of the mechanisms how HCV disrupts the innate immunity and causes persistent infection in hepatocytes.